Advances in cellular and molecular biology have made it possible, in certain cases, to identify a gene encoding a desired protein, to isolate the gene, to insert the gene into a host cell and to express the inserted gene in the host cell to produce the desired protein. Bacteria, especially Escherichia coli and Bacillus subtilis, have been intensively studied as host cells. When bacteria are used as host cells for this heterologous gene expression, however, two problems frequently have been encountered. Most bacterial expression systems produce proteins intracellularly. When high level expression is achieved, the protein is often found to be insoluble (Marston et al., 1984; Williams et al.; 1982; Schoner et al., 1985). Production of an active protein from this insoluble material requires solubilization and refolding protocols which are often prohibitively expensive. If the protein is produced in an active, soluble form within the cell, its isolation requires cell lysis which releases hundreds of other soluble intracellular proteins. This can present a formidable problem in purification of the desired product.
Both the problem of production of insoluble, inactive proteins and of difficulty of purification may be overcome by having the bacteria secrete the desired protein into the growth medium. One particularly well documented method of directing the secretion of proteins is the use of a secretory signal sequence (Randall and Hardy, 1984; Silhavy et al., 1983; Wickner, 1979). When a signal peptide is fused to the amino-terminal end of a heterologous protein, it directs the heterologous protein to the secretory machinery at the cell membrane. The heterologous protein is then translocated across the membrane and a specific protease, sometimes referred to as "signal peptidase," removes the signal peptide and releases the heterologous protein. In E. coli, secretion results in the accumulation of the heterologous protein in the periplasmic space, while in a gram positive bacterium, such as B. subtilis, secretion results in the accumulation of the product in the culture medium. This method has been used to direct the secretion of heterologous proteins in bacteria (Fraser and Bruce, 1978; Palva et al., 1983; Talmadge et al., 1981). As a result of these and other studies, problems, both potential and realized, have been discovered in the use of this particular approach. Cleavage of the signal peptide by signal peptidase may not be efficient or even accurate. Consequently, the secreted population of heterologous protein may contain unprocessed or misprocessed subpopulations. In addition, the amount of heterologous protein secreted is usually very small, and because both E. coli and B. subtilis also secrete proteases, a significant amount of the heterologous protein can be degraded after it is secreted.
Because of this latter point, the secretion and accumulation of heterologous proteins in the culture medium by B. subtilis is vitiated unless the host cell expression and secretion of proteolytic enzymes is minimized or eliminated. One method for minimizing the effect of protease degradation of secreted proteins is to utilize mutant strains deficient in protease production. Mutations have been isolated in both the alkaline and neutral protease structural genes by recombinant methods (Stahl and Ferrari, 1984; Yang et al., 1984; Kawamura and Doi, 1984). Other protease deficient mutations isolated, to date, are pleitropic and also block the formation of mature endospores (Michel and Millet, 1970). Many of these mutations cause the cells to lyse when the culture is in the stationary phase of growth, thus may not be desirable for use in B. subtilis for the expression and secretion of heterologous proteins. While the use of existing protease deficient mutants may reduce the problem of product instability, it may be necessary to isolate mutations in other protease genes to obtain maximum product stability.
In addition to using mutants of B. subtilis, the onset of endospore development and the secretion of proteases can be reduced significantly simply by adding to the medium a substance, such as glucose, which blocks the onset of secondary metabolism (Hoch, 1976). In the presence of glucose, the secretion of many proteases and cell lysis are inhibited. Cell lysis is to be avoided since release of intracellular proteins, of which some could be proteases, could result in additional degradation of the product and make it more difficult and costly to purify.
We have now discovered a new method for microbial production and export of a desired protein which avoids some of the problems associated with secretion via a signal peptide and secretion during stationary phase of growth. The method of this invention results in the transport of protein out of a flagellated bacterium and does so during the logarithmic growth phase and in the presence of a repressive substance such as glucose. Products thus secreted are likely to be spared the problem of degradation by some proteases. Combining this secretion method with protease deficient mutants may improve product stability even more. This method harnesses the export system normally used by the host cell in exporting the protein flagellin.
Before describing the subject invention in detail, it may be helpful to set forth briefly further background information concerning flagellin.
Flagellin, which is the monomeric protein component of the flgellar filament, is a major extracellular protein product in many bacteria. Specifically, it is the predominant extracellular protein in logarithmic and early stationary phase of growth when Bacillus is grown in minimal salts and glucose. The mechanism by which flagellin is exported is unknown. It does not seem to be exported by using a signal sequence which is cleaved from the amino-terminus of the protein (Silhavy et al., 1983). The amino-terminus of purified flagellin from Caulobacter crescentus, for example, has a sequence which corresponds to the putative translation start of its cloned structural gene (Gill and Aggbian, 1982, 1983). The amino-terminus of purified flagellin from Salmonella typhimurium begins with alanine which corresponds to the second amino acid following the translation start of its cloned structural gene (Joys and Rankis, 1972; Zieg and Simon, 1980). It is therefore unlikely that a processed leader sequence mediates transport of flagellin in bacteria such as Bacillus, Salmonella or Caulobacter.
Flagellin and several other proteins seem to exit the cell through the central core of the flagellum (lino, 1977; Silverman and Simon, 1977). These proteins can be as large as about 60 Kd so the physical size of the organelle core does not seem to limit this system unduly. The mechanism of secretion and the structural necessities of proteins to be exported by this system are not known, but much information about this system and the related system in E. coli has been collected and reviewed by lino (1977) and Silverman and Simon (1977). One notable feature of the system is its efficiency. It suffices to note that a flagellated E. coli cell has some 60,000 flagellin molecules (Komeda, 1982), thus a culture containing 1.times.10.sup.9 cells per ml exports approximately 5 mg per liter of flagellin.
To date, a minimum of 40 genes have been identified in E. coli which are apparently involved in bacterial motility and 29 involved in the synthesis of the flagellar organelle (lino, 1977; Silverman and Simon, 1977). A pathway for the assembly of a flagellum was proposed by Suzuki and Komeda (1981). The central dogma in flagellar assembly is that the structure is assembled from the cell membrane outward and the new components are derived from proteins that are transported through the core of the organelle and are assembled on the tip of the growing organelle. The flagellin structural gene is one of the last flagellar genes to be transcribed and translated during the synthesis of the flagellar organelle. Thus, a strain deleted for the flagellin gene should have an intact basal body and hook structure but would lack the filament. A mutation of interest to this invention is the cfs mutation, which has a phenotype of constitutive flagellar synthesis when this strain is grown in the presence of glucose (Silverman and Simon, 1977). E. coli strains carrying this particular mutation also produce five-fold mre flagellin than wild-type strains.
Grant and Simon, (1969), isolated temperature sensitive (ts)fla mutants of B. subtilis 1968 by isolating mutants resistant to bacteriophage PBS1 at high but now at lower temperatures. To date, 3 alleles of the hag locus (encoding the so-called "h-antigen" which is the flagellin protein) in B. subtilis have been described. Wild-type B. subtilis 168 contains the hag-1 allele, B. subtilis W23 has hag-2, and hag-3 is a "straight" mutant of hag-1. Another mutation of interest to this invention is the ifm mutation, which has a phenotype of higher motility and increased flagellin production (Grant and Simon, 1969; Pooley and Karamata, 1984).
In reducing to practice the present invention we have isolated and determined the sequence of the B. subtilis hag gene; deleted, in certain embodiments, part or all of this gene from the genome of the host cell; identified essential elements of the sequence involved in transport of the protein to the outside of the cell; inserted into the host cell a heterologous gene encoding a desired protein at some site within the genome of the bacterium or within a flagellin gene locus of the host cell genome or as an extrachromosomal plasmid and expressed and exported fusion proteins containing the desired protein fused to that portion of flagellin essential for export. Methods and materials for the execution of this strategy are disclosed in detail hereinafter.